Detection and amplification of nucleic acids play important roles in genetic analysis, molecular diagnostics, and drug discovery. Many such applications require specific, sensitive and inexpensive quantitative detection of certain DNA or RNA molecules, gene expression, DNA mutations or DNA methylation present in a small fraction of total polynucleotides. Many current methods use polymerase chain reaction, or PCR, and specifically, real-time PCR (quantitative, or qPCR) to detect and quantify very small amounts of DNA or RNA from clinical samples.
While the performance of current PCR assays is constantly improving, their sensitivity, specificity and cost are still far away from becoming a widely acceptable diagnostic test. Indeed, many PCR methods currently used in the art suffer from technical limitations that make the methods inadequate for many practical applications. For example, in instances where the target molecule has secondary structure that inhibits or even completely prevents binding of one or both primers to the target, amplification can be reduced or even non-existent, which, for example, from a diagnostic standpoint could give rise to a false negative despite use of a highly specific primer with binding properties that would be expected to be sensitive. Other challenges include low sensitivity of current real-time PCR assays in detection and discrimination of rare DNA molecules with a single base mutation in situations when they mixed with thousands of non-mutated DNA molecules, and ability to combine multiple mutation detection assays into one multiplex diagnostic assay.
There thus remains a need in the art for a development of amplification primers that combines high binding specificity with low synthesis cost that retain the ability to overcome technical problems recognized in the art, including novel application of PCR for diagnostics using next generation sequencing platforms.